Protocol for the isolation of mouse senescence-associated CD4+ T cells using flow cytometry and functional assays.

Senescence-associated (SA) CD4+ T cells, which increase with age, may underlie the development of autoimmunity and chronic inflammation, but their pathological function remains understudied. Here, we present a protocol to isolate CD153+ SA-T cells and evaluate their characteristic responses upon T cell receptor stimulation. We describe steps for the isolation of CD153+ SA-T cells using flow cytometry and in vitro culture with stimulatory antibodies against CD3, CD28, and CD153. We then detail the assessment of the proliferation capacity and cytokine production. For complete details on the use and execution of this protocol, please refer to Fukushima et al. (2022).1.

cis interaction of CD153 with TCR/CD3 is crucial for the pathogenic activation of senescence-associated T cells.

With age, senescence-associated (SA) CD4+ T cells that are refractory to T cell receptor (TCR) stimulation are increased along with spontaneous germinal center (Spt-GC) development prone to autoantibody production. We demonstrate that CD153 and its receptor CD30 are expressed in SA-T and Spt-GC B cells, respectively, and deficiency of either CD153 or CD30 results in the compromised increase of both cell types. CD153 engagement on SA-T cells upon TCR stimulation causes association of CD153 with the TCR/CD3 complex and restores TCR signaling, whereas CD30 engagement on GC B cells induces their expansion. Administration of an anti-CD153 antibody blocking the interaction with CD30 suppresses the increase in both SA-T and Spt-GC B cells with age and ameliorates lupus in lupus-prone mice. These results suggest that the molecular interaction of CD153 and CD30 plays a central role in the reciprocal activation of SA-T and Spt-GC B cells, leading to immunosenescent phenotypes and autoimmunity.

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury.

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

The impact of senescence-associated T cells on immunosenescence and age-related disorders.

Immunosenescence is age-associated changes in the immunological functions, including diminished acquired immunity against infection, pro-inflammatory traits, and increased risk of autoimmunity. The proportions of memory-phenotype T cells in the peripheral T cell population steadily increase with age, but the relationship between this change and immunosenescent phenotypes remains elusive. Recently, we identified a minor memory-phenotype CD4+ T cell subpopulation that constitutively expressed PD-1 and CD153 as a bona fide age-dependent T cell population; we termed these cells senescence-associated T (SA-T) cells. SA-T cells exhibit characteristic features of cellular senescence, with defective T cell receptor-mediated proliferation and T cell cytokine production. However, upon T cell receptor stimulation, SA-T cells secrete abundant atypical pro-inflammatory cytokines such as osteopontin and chemokines, reminiscent of the SA-secretory phenotype. In addition to aging, SA-T cells accumulate and cause persistent inflammation in tissues following a wide range of insults including immune complex deposition, metabolic stresses, vascular damages, and tumors. In this review, we summarize the recent understanding of immunosenescence with particular focus on SA-T cells and their role in various age-related disorders.

The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine.

Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. However, cellular activation during disease development induces Opn expression in various immune cells. In this study, using Opn-EGFP knock-in (KI) mice we found that CD8α+ T cells in the intestinal tissues, including Peyer's patch, lamina propria and epithelium, express Opn under steady state conditions. Therefore, we examined the role of Opn-expressing CD8α+ T cells in intestinal homeostasis. Interestingly, Opn knockout (KO) mice had altered fecal microflora concordant with a reduction of TCRγδ+ intraepithelial lymphocytes (IELs). Consistent with this result, both treatment with anti-Opn blocking antibody and deficiency of Opn resulted in decreased survival of TCRγδ+ and TCRαß+ IELs. This data suggests that a possibility that Opn may function as a survival factor for IELs in the intestinal tissue. Collectively, these data suggest the possibility that Opn might regulate the homeostasis of intestinal microflora through maintenance of TCRγδ+ IELs, possibly by support of IEL survival.

Osteopontin in Spontaneous Germinal Centers Inhibits Apoptotic Cell Engulfment and Promotes Anti-Nuclear Antibody Production in Lupus-Prone Mice.

Disposal of apoptotic cells is important for tissue homeostasis. Defects in this process in immune tissues may lead to breakdown of self-tolerance against intracellular molecules, including nuclear components. Development of diverse anti-nuclear Abs (ANAs) is a hallmark of lupus, which may arise, in part, due to impaired apoptotic cell clearance. In this work, we demonstrate that spontaneous germinal centers (GCs) in lupus-prone mice contain significantly elevated levels of unengulfed apoptotic cells, which are otherwise swiftly engulfed by tingible body macrophages. We indicate that osteopontin (OPN) secreted by CD153(+) senescence-associated T cells, which selectively accumulate in the GCs of lupus-prone mice, interferes with phagocytosis of apoptotic cells specifically captured via MFG-E8. OPN induced diffuse and prolonged Rac1 activation in phagocytes via integrin αvß3 and inhibited the dissolution of phagocytic actin cup, causing defective apoptotic cell engulfment. In wild-type B6 mice, administration of TLR7 ligand also caused spontaneous GC reactions with increasing unengulfed apoptotic cells and ANA production, whereas B6 mice deficient for Spp1 encoding OPN showed less apoptotic cells and developed significantly reduced ANAs in response to TLR7 ligand. Our results suggest that OPN secreted by follicular CD153(+) senescence-associated T cells in GCs promotes a continuous supply of intracellular autoantigens via apoptotic cells, thus playing a key role in the progression of the autoreactive GC reaction and leading to pathogenic autoantibody production in lupus-prone mice.

A CD153+CD4+ T follicular cell population with cell-senescence features plays a crucial role in lupus pathogenesis via osteopontin production.

Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.

Anesthesia with propofol induces insulin resistance systemically in skeletal and cardiac muscles and liver of rats.

Hyperglycemia together with hepatic and muscle insulin resistance are common features in critically ill patients, and these changes are associated with enhanced inflammatory response, increased susceptibility to infection, muscle wasting, and worsened prognosis. Tight blood glucose control by intensive insulin treatment may reduce the morbidity and mortality in intensive care units. Although some anesthetics have been shown to cause insulin resistance, it remains unknown how and in which tissues insulin resistance is induced by anesthetics. Moreover, the effects of propofol, a clinically relevant intravenous anesthetic, also used in the intensive care unit for sedation, on insulin sensitivity have not yet been investigated. Euglycemic hyperinsulinemic clamp study was performed in rats anesthetized with propofol and conscious unrestrained rats. To evaluate glucose uptake in tissues and hepatic glucose output [(3)H]glucose and 2-deoxy[(14)C]glucose were infused during the clamp study. Anesthesia with propofol induced a marked whole-body insulin resistance compared with conscious rats, as reflected by significantly decreased glucose infusion rate to maintain euglycemia. Insulin-stimulated tissue glucose uptake was decreased in skeletal muscle and heart, and hepatic glucose output was increased in propofol anesthetized rats. Anesthesia with propofol induces systemic insulin resistance along with decreases in insulin-stimulated glucose uptake in skeletal and heart muscle and attenuation of the insulin-mediated suppression of hepatic glucose output in rats.

iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3ß activation in skeletal muscle of rats.

OBJECTIVES: Recent studies suggest that activation of glycogen synthase kinase (GSK)-3ß may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3ß activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3ß activity in skeletal muscle of burned rats. MATERIALS/METHODS: Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3ß activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3ß and its endogenous substrate, glycogen synthase. RESULTS: GSK-3ß activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3ß activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3ß activity in sham-burned rats. CONCLUSIONS: Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3ß activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3ß.

[Effect on fetal umbilical arterial blood of administration of vasopressors for hypotension after spinal anesthesia during cesarean section].

Spinal anesthesia was performed in 40 patients undergoing cesarean section. When systolic blood pressure dropped below 100 mmHg, phenylephrine 100-200 microg (P group) or ephedrine 5-10 mg (E group) was administered. The pH of the umbilical arterial blood was collected after delivery of the baby. Apgar scores, and maternal systolic blood pressure and heart rate before and after each drug administration were compared retrospectively. The umbilical arterial pH and Apgar scores tended to be slightly higher in the P group, but there was no significant difference between the two groups. The rate of blood pressure elevation was 27% in the P group and 41% in the E group. The heart rate decreased significantly in the P group. There was no significant difference in the systolic blood pressure before administration of each drug. Recently, it is reported that the umbilical arterial pH is higher in cases in which phenylephrine is used for hypotension after spinal anesthesia during a cesarean section. However, the optimal dose of phenylephrine is debatable and has not been established. More studies are necessary to determine which drugs should be selected according to the maternal condition.

Molecular cloning of chicken interleukin-5 receptor α-chain and analysis of its binding specificity.

Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41-43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.

Induction of immune suppression in the chick by an optimal dose of an immunizing antigen in the presence of its specific maternal antibody.

Prolonged interference or suppression of maternal antibodies of the humoral immune response of newly hatched chicks to active immunization has been documented; however, the immunological mechanisms responsible for such suppression are still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specific IgY antibodies were transferred by yolk sac inoculation to newly hatched chicks, and they were immunized with DNP-KLH or rabbit serum albumen (RSA) at 1 and 4 weeks of age. The concentrations of anti-DNP and anti-RSA antibodies in serum samples of these chicks were measured using an enzyme-linked immunosorbent assay (ELISA). The immune responses of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with an appropriate dose of DNP-KLH were suppressed. However, those of the chicks that received the same high dose of maternal non-specific IgY antibodies and were immunized with an appropriate dose of DNP-KLH and those of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with RSA were not suppressed. On the other hand, suppression of anti-DNP antibody production would not be induced if the chicks received a high dose of antigen specific maternal antibodies and were immunized with a high dose of the same antigen. These results revealed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depended mainly on the ratio of antigen/maternal antibody at the time of immunization.

[Anesthetic management for a patient with anti-phospholipid antigen syndrome].

A 54-year-old man with anti-phospholipid antigen syndrome (APS) was to undergo artifitial vascular replacement for arteriosclerotic obliterans. APS is characterized by the presense of anti-phospholipid antibodies, hypercoagulability, and prolonged phospholipid dependent coagulation such as activated clotting time (ACT). Perioperative thrombotic complications are frequent among patients with anti-phospholipid antigen syndrome. In these cases, perioperative heparin titration for anticoagulation monitoring is very important. In this patient, preoperative coagulation testing revealed prolonged partial thromboplastin time (APTT) and antithrombin-III (AT-III). In the operating room, a base line ACT was 124 sec. We decided to administer a standard protocol heparine dose of 100 IU x kg(-1) of body weight. After the artifitial vascular replacement, the ACT was recorded at 249 sec. Therefore, a 75 mg dose of protamine was administered. In this particular case, it is difficult to decided to administer the heparin dose. When an adequate platelet count, normal bleeding time, normal TT, and normal AT-III level are shown, it would be decided to use the heparin monitoring systems; ACT, circulating heparin concentrations, according to some reports. In this case, with standard protocol heparin, he had no complications in the postoperative days. The perioperative management was succeseful.

Prolonged suppression of chick humoral immune response by antigen specific maternal antibody.

Although the inhibitory effect of maternal antibodies on active immunization of neonates has been extensively documented, much less attention has been devoted on the exact level of these antibodies which can induce this effect and the extent of such effect. Firstly, laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH).Then, maternal anti-DNP antibodies in chicks derived from these hens were measured by using enzyme-linked immunosorbent assay (ELISA). Chicks with high levels of maternal anti-DNP showed immune suppression, while chicks with low levels of maternal anti-DNP showed normal immune response when they immunized with the same antigen at 1 and 4 weeks of age. Then, different doses of purified maternal anti-DNP were transferred to fertile eggs at 16 days of embryogenesis by in ovo injection and all chicks were immunized with DNP-KLH at 1 and 4 weeks of age. Chicks received 1 mg of anti-DNP showed normal immune response, chicks received 3 mg of anti-DNP showed weak immune response, and chicks received 5 and 8 mg of anti-DNP showed immune suppression. Chicks received 8 mg of anti-DNP were immunized with DNP-KLH at 4 and 7 weeks of age. Their immune response was significantly lower than that of chicks of no-maternal anti-DNP. These results suggested that high levels of maternal antibodies interfere or suppress the immune response of active immunization not only at early period but also at the period in which the maternal antibodies at very low levels.

[Phase I study of intrathecal chemotherapy with NS-101 for leptomeningeal carcinomatosis in Japan].

Intrathecal administration of MTX and/or Ara-C through the Ommaya system placed in a ventricle has been accepted as a standard therapy for leptomeningeal carcinomatosis. Recently, sustained-release cytosine arabinoside, a depot cytosine arabinoside liposomal injection: DepoCyt, has been reported to be useful for the treatment of leptomeningeal carcinomatosis in Canada, Europe, and the USA. Phase I study of NS-101, DepoCyt in Japan, was performed in patients with leptomeningeal carcinomatosis from solid tumors using the continuous reassessment method (CRM). NS-101 was administered twice through the Ommaya system placed in the lateral ventricle with an interval of 2 weeks. A dose of 25 mg was initially tried in a patient and then a dose of 50 mg was administered in 8 patients. The recommended dose of NS-101 was estimated from pharmacodynamics and tolerance from CRM, and the clinical effectiveness of NS-101 was also assessed. We also compared the present data with the reported data from Europe and the USA. As a result, free Ara-C was maintained over the estimated effective concentration for 2 weeks, and no pharmacodynamic differences were confirmed among Japan, Europe and the USA. Maximum tolerated dose was determined to be 50 mg. Complete cytological response of cerebrospinal fluid (CSF) was obtained in a patient, and CSF cytological conversion of either ventricle or lumbar CSF from positive to negative was observed in four patients. Neurological improvements in 3 pts and no sign of worsening in one patient were observed, and grade IV toxicity based on National Cancer Institute-Common Toxicity Criteria was not encountered. In conclusion, NS-101 was effective for leptomeningeal carcinomatosis from solid tumors and the estimated maximum tolerated dose was 50 mg as approved in Europe and the USA.

Induction of glioma cell migration by vitronectin in human serum and cerebrospinal fluid.

OBJECT: Malignant gliomas are often highly invasive and can migrate along blood vessels. The purpose of the current study was to identify the substance in human serum and/or cerebrospinal fluid (CSF) that promotes glioma cell migration. METHODS: The authors used a Boyden chamber cell migration assay to study the effect of serum from patients with glioma and healthy volunteers on chemotaxis of A172 human glioma cells. Heat inactivation, trypsinization, and ultrafiltration of serum were used to establish the nature of the active factor. Vitronectin and fibronectin were chosen for further investigations; chemotactic effects were studied in both serum and CSF. RESULTS: Serum from both patients with glioma and healthy volunteers was found to promote chemotaxis of human glioma cells. This activity was greatly reduced by heat inactivation or trypsinization. Fractionation of the serum by ultrafiltration through membranes with various pore sizes showed that the active molecule was larger than 50 kD. Antibodies against integrin alphav or alphavbeta5 or arginine-glycine-aspartic acid-containing peptides, both of which block the vitronectin-glioma cell interactions, significantly reduced serum-induced cell migration, whereas blocking the interaction of glioma cells with fibronectin had no effect. Furthermore, the ability of serum to promote the migration of A 172 or T98G glioma cells was suppressed by immunodepletion of vitronectin and restored by the addition of exogenous vitronectin. The migration of glioma cells induced by CSF collected from the postoperative cavity of a malignant glioma patient was also reduced by blocking the interaction of glioma cells with vitronectin. CONCLUSIONS: These results suggest that vitronectin is one of the major factors in serum- and CSF-induced glioma cell migration.

Cloning of the chicken interleukin-13 receptor alpha 2 gene and production of a specific monoclonal antibody.

Mammalian interleukin-13 (IL-13) is an important regulatory T2 cytokine secreted by activated T lymphocytes. The IL-13 receptor (IL-13R) has two different chains, IL-13Ralpha1 and IL-13Ralpha2. Although the chicken IL-13 gene is well characterized, little is known about IL-13Rs. We cloned a cDNA encoding the 380 amino acid pro-peptide of chicken IL-13Ralpha2 (chIL-13Ralpha2) and developed a monoclonal antibody (mAb), HU13-1, against it. The chIL-13Ralpha2 amino acid sequence showed 37-39% sequence identity with mammalian homologs. High levels of chIL-13Ralpha2 mRNA were expressed in liver, testis, ovary, brain, and lipopolysaccharide (LPS)-stimulated IN24 cells. HU13-1 specifically recognized recombinant chIL-13Ralpha2 in ELISAs, and western blots identified a 45-kDa glycoprotein or a 41-kDa non-glycosylated protein in LPS-stimulated IN24 cell lysates. LPS induced a gradual increase in HU13-1-positive IN24 cells over 20 h. These results indicate that mAb HU13-1 recognizes native chIL-13Ralpha2 and will be valuable for further studies of chIL-13Rs.

Intrathecal administration of Y-27632, a specific rho-associated kinase inhibitor, for rat neoplastic meningitis.

The small GTP-binding protein Rho and its target Rho-associated kinase trigger an intracellular signaling cascade that controls actin cytoskeleton and plays an essential role in cell motility and adhesion. A specific Rho-associated kinase inhibitor, Y-27632, has been reported to inhibit cancer invasion. Clinically, disseminated tumor cells in the cerebrospinal fluid invade the intraparenchymal region, damaging the brain and nerves, resulting in fatal brain stem dysfunction, despite intrathecal chemotherapy. To expand therapeutic options for this devastating neoplastic meningitis, we evaluated the potential use of intrathecal Y-27632 administration by employing Walker 256 cells, a rat mammary cancer cell line. Y-27632 dose-dependently inhibited chemotactic and invasive activity of Walker 256 cells. Y-27632 also inhibited the phosphorylation level of regulatory myosin light chain in vitro, but the effect was temporary and was considerably diminished within 16 hours. Y-27632 induced striking morphologic changes in Walker 256 cells, as evidenced by decreased cell-matrix adhesion in culture dishes and three-dimensional collagen I gels, and slightly inhibited colony formation in soft agar. Nevertheless, this drug treatment did not affect Walker 256 cell growth rate. We were able to administer continuous delivery of this inhibitor using an osmotic pump and maintaining drug concentration of 10 mumol/L within the brain. Importantly, this concentration of Y-27632 showed minimal neurotoxicity both in vitro and in vivo. We found that an intrathecal therapy, combining 5-fluoro-2'-deoxyuridine with Y-27632, significantly increased the survival time of rats bearing meningeal carcinomatosis in comparison with animals treated with 5-fluoro-2'-deoxyuridine alone. Taken together, our findings indicate that continuous intrathecal administration of Y-27632 could be a promising therapeutic method when combined with chemotherapy for treating human neoplastic meningitis.

Continuous intrathecal administration of 5-fluoro-2'-deoxyuridine for the treatment of neoplastic meningitis.

OBJECTIVE: Previously, we reported a good clinical treatment effect of intrathecal chemotherapy by repeated bolus administration of 5-fluoro-2'-deoxyuridine (FdUrd) for neoplastic meningitis (NM). Moreover, we detected no side effects or neurotoxicity despite the long-term repetition of intrathecal administration. On the basis of these findings, continuous intrathecal chemotherapy (CIC) with FdUrd for patients with NM was attempted using a simple pump system. We evaluated the usefulness of CIC with FdUrd for the treatment of NM. METHODS: A total of 25 patients were enrolled in this study. FdUrd (1.0 mg/d) was administered using a balloon pump system. CIC was continued as long as possible. Eight patients received whole-brain irradiation (3 Gy x 10) simultaneously with CIC. The effects of the treatment were analyzed in terms of improvement in neurological signs and symptoms and the findings of ventricular and lumbar cerebrospinal fluid analysis 2 and 4 weeks after CIC was initiated and on magnetic resonance imaging scans 2 months after CIC began. RESULTS: No apparent toxicity has been observed to date. Evidence of a cerebrospinal fluid response was observed in 13 patients. Headache and nausea were improved in all patients, and cranial nerve impairment was improved in 12 patients. A magnetic resonance imaging response was observed in only 5 patients. Overall response was observed in 15 patients when cases of stable disease were excluded from the responding cases. Survival time from the commencement of CIC (mean +/- standard error of the mean) was 255 +/- 30 days in 25 patients. CONCLUSION: This therapy may be useful, especially as a maintenance therapy for NM.